The human genome is composed of approximately 4x10/9 base pairs of DNA. In order to grow, a human cell must precisely duplicate this entire genome during each cell cycle. This requires the concerted action of many proteins to initiate DNA replication specifically to multiple origins and to synthesize large amounts of DNA accurately. The goal of this research is to elucidate the molecular mechanism(s) of eukaryotic DNA replication and how DNA replication is regulated in the cell. This research focuses on Replication Protein A (RPA), a multi-subunit protein that binds to single-stranded. This human protein is absolutely required for DNA replication, DNA repair and recombination. In addition to binding to single-stranded DNA, RPA interacts with regulation (e.g. p53). These interactions appear to be required for RPA function. RPA also become phosphorylated appears to regulated RPA-protein interactions. The goal of these studies is to elucidate the role of RPA in DNA replication and other reactions of cellular metabolism. A combination of mutational analyses, direct biochemical assays, replication assays and genetic analysis will be used to: (1) define the molecular interactions of RNA with DNA, (2) elucidate the roles of RNA in DNA replication and determine the role(s) of RPA in cellular coordination of DNA metabolism and (3) determine the structure of RPA and characterize conformation changes in RPA. These studies will lead to a better understanding of the molecular mechanism of DNA replication and cellular DNA metabolism.